Canine distemper virus DNA vaccination of mink can overcome interference by maternal antibodies.

Canine distemper virus (CDV) is highly contagious and can cause severe disease against which conventional live vaccines are ineffective in the presence of maternal antibodies. Vaccination in the presences of maternal antibodies was challenged by vaccination of 5 days old and 3 weeks old mink kits with CDV DNA vaccines. Virus neutralising (VN) antibody responses were induced in mink kits vaccinated with a plasmid encoding the haemaglutinin protein (H) of CDV (n=5, pCDV-H) or a combination of the H, fusion (F) and nucleoprotein (N) of CDV (n=5, pCDV-HFN). These DNA vaccinated kits were protected against virulent experimental infection with field strains of CDV. The pCDV-H was more efficient in inducing protective immunity in the presence of maternal antibodies compared to the pCDV-HFN. The results show that DNA vaccination with the pCDV-H or pCDV-HFN (n=4) only given once at 5 days of age induces virus specific immune response in neonatal mink and protection against virulent CDV exposure later in life.

Carbohydrate determinants in ferret conjunctiva are affected by infection with influenza H1N1 virus.

BACKGROUND: Carbohydrates often accomplish as cell-surface receptors for microorganisms and influenza virus preferentially binds to sialic acid through the viral haemagglutinin. The virus may attach not only to the epithelium in the airways, but also to the surface ocular epithelium. PURPOSE: To decide if ferrets can be used to study virus induced conjunctivitis and to evaluate changes in the conjunctival glycosylation pattern during an influenza attack. METHODS: Ferrets were infected with H1N1 influenza virus via nasal inoculation. The in situ carbohydrate expressions in eyelid sections from ferrets 0 to 10 days after infection was examined using lectin- and immunohistochemistry. RESULTS: The conjunctival cells became hypertrophic with appearance of both PAS positive and PAS + Alcian Blue stained cells 5-6 days after inoculation. The binding of three sialic acid detecting lectins were investigated: WGA, MAA2 and SNA1. While none of them stained conjunctival epithelial cells in the non-infected ferrets to any extent, there was a positive conjunctival reaction in the infected ferret after incubation with all three lectins. Binding of a MUC1 antibody that seems to detect sialylated determinants in the mucin molecule indicates that MUC1 is de novo expressed in most of the squamous conjunctival cells at the start of the influenza infection. MUC5AC positive epithelial cells, probably goblet cells, proliferate in the diseased conjunctiva. CONCLUSION: Nasal inoculation of H1N1 virus to ferrets has an effect on the conjunctival cells and change their expression of glycans. Synthesized glycans are an integral part of the tear film and the present study contributes to reveal the changes that occur in the surface epithelium in the eyelid and thereby to elucidate the pathophysiology of the virus mediated conjunctivitis. Ferrets are suitable animal models to study human conjunctivitis mediated by human influenza virus.

DNA vaccines encoding proteins from wild-type and attenuated canine distemper virus protect equally well against wild-type virus challenge.

Immunity induced by DNA vaccines containing the hemagglutinin (H) and nucleoprotein (N) genes of wild-type and attenuated canine distemper virus (CDV) was investigated in mink (Mustela vison), a highly susceptible natural host of CDV. All DNA-immunized mink seroconverted, and significant levels of virus-neutralizing (VN) antibodies were present on the day of challenge with wild-type CDV. The DNA vaccines also primed the cell-mediated memory responses, as indicated by an early increase in the number of interferon-gamma (IFN-γ)-producing lymphocytes after challenge. Importantly, the wild-type and attenuated CDV DNA vaccines had a long-term protective effect against wild-type CDV challenge. The vaccine-induced immunity induced by the H and N genes from wild-type CDV and those from attenuated CDV was comparable. Because these two DNA vaccines were shown to protect equally well against wild-type virus challenge, it is suggested that the genetic/antigenic heterogeneity between vaccine strains and contemporary wild-type strains are unlikely to cause vaccine failure.

Manipulation of the gut microbiota in C57BL/6 mice changes glucose tolerance without affecting weight development and gut mucosal immunity.

Inflammatory diseases such as type 2 diabetes (T2D) in humans and mice are under the influence of the composition of the gut microbiota (GM). It was previously demonstrated that treating Lep(ob) mice with antibiotics improved glucose tolerance. However, wild type C57BL/6J mice may also exhibit plasma glucose intolerance reminiscent of human T2D. We hypothesized that antibiotic treatment in C57BL/6 mice would have an impact on glucose tolerance without affecting weight and gut immunology. When compared to mice treated with erythromycin or the controls, treatment for five weeks with ampicillin improved glucose tolerance without significantly affecting the weight or the number of gut mucosal regulatory T cells, tolerogenic dendritic cells or T helper cells type 1. 16S rRNA gene based denaturing gradient gel electrophoresis profiles clearly clustered according to treatment and showed that antibiotic treatment reduced GM diversity. It is concluded that antibiotic treatment changes glucose metabolism as well as the composition of the GM in C57BL/6 mice, and that this does not seem to be correlated to weight development in the mice.

CAF01 potentiates immune responses and efficacy of an inactivated influenza vaccine in ferrets.

Trivalent inactivated vaccines (TIV) against influenza are given to 350 million people every year. Most of these are non-adjuvanted vaccines whose immunogenicity and protective efficacy are considered suboptimal. Commercially available non-adjuvanted TIV are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (dimethyldioctadecylammonium/trehalose 6,6'-dibehenate, CAF01) was developed. CAF01 has proven to enhance both humoral and cell-mediated immune responses to a number of different experimental vaccine candidates. In this study, we compared the immune responses in ferrets to a commercially available TIV with the responses to the same vaccine mixed with the CAF01 adjuvant. Two recently circulating H1N1 viruses were used as challenge to test the vaccine efficacy. CAF01 improved the immunogenicity of the vaccine, with increased influenza-specific IgA and IgG levels. Additionally, CAF01 promoted cellular-mediated immunity as indicated by interferon-gamma expressing lymphocytes, measured by flow cytometry. CAF01 also enhanced the protection conferred by the vaccine by reducing the viral load measured in nasal washes by RT-PCR. Finally, CAF01 allowed for dose-reduction and led to higher levels of protection compared to TIV adjuvanted with a squalene emulsion. The data obtained in this human-relevant challenge model supports the potential of CAF01 in future influenza vaccines.

Alcohol facilitates CD1d loading, subsequent activation of NKT cells, and reduces the incidence of diabetes in NOD mice.

BACKGROUND: Ethanol ('alcohol') is a partly hydrophobic detergent that may affect the accessibility of glycolipids thereby influencing immunological effects of these molecules. METHODS: The study included cellular in vitro tests using α-galactosylceramide (αGalCer), and in vivo NOD mice experiments detecting diabetes incidence and performing behavioural and bacterial analyses. RESULTS: Alcohol in concentrations from 0.6% to 2.5% increased IL-2 production from NKT cells stimulated with αGalCer by 60% (p<0.05). CD1d expressed on HeLa cells contained significantly increasing amounts of αGalCer with increasing concentrations of alcohol, suggesting that alcohol facilitated the passive loading of αGalCer to CD1d. NOD mice were found to tolerate 5% ethanol in their drinking water without signs of impairment in liver function. Giving this treatment, the diabetes incidence declined significantly. Higher numbers of CD3+CD49b+ NKT cells were found in spleen and liver of the alcohol treated compared to the control mice (p<0.05), whereas the amount of CD4+Foxp3+ regulator T cells did not differ. Increased concentrations of IFN-γ were detected in 24-hour blood samples of alcohol treated mice. Behavioural studies showed no change in attitude of the ethanol-consuming mice, and bacterial composition of caecum samples was not affected by alcohol, disqualifying these as protective mechanisms. CONCLUSION: Alcohol facilitates the uptake of glycolipids and the stimulation of NKT cells, which are known to counteract Type 1 diabetes development. We propose that this is the acting mechanism by which treatment with alcohol reduces the incidence of diabetes in NOD mice. This is corroborated by epidemiology showing beneficial effect of alcohol to reduce the severity of atherosclerosis and related diseases.

Pandemic influenza 1918 H1N1 and 1968 H3N2 DNA vaccines induce cross-reactive immunity in ferrets against infection with viruses drifted for decades.

BACKGROUND: Alternative influenza vaccines and vaccine production forms are needed as the conventional protein vaccines do not induce broad cross-reactivity against drifted strains. Furthermore, fast vaccine production is especially important in a pandemic situation, and broader vaccine reactivity would diminish the need for frequent change in the vaccine formulations. OBJECTIVE: In this study, we compared the ability of pandemic influenza DNA vaccines to induce immunity against distantly related strains within a subtype with the immunity induced by conventional trivalent protein vaccines against homologous virus challenge. METHODS: Ferrets were immunised by particle-mediated epidermal delivery (gene gun) with DNA vaccines based on the haemagglutinin (HA) and neuraminidase (NA) and/or the matrix (M) and nucleoprotein genes of the 1918 H1N1 Spanish influenza pandemic virus or the 1968 H3N2 Hong Kong influenza pandemic virus. The animals were challenged with contemporary H1N1 or H3N2 viruses. RESULTS: We demonstrated that DNA vaccines encoding proteins of the original 1918 H1N1 pandemic virus induced protective cross-reactive immune responses in ferrets against infection with a 1947 H1N1 virus and a recent 1999 H1N1 virus. Similarly, a DNA vaccine, based on the HA and NA of the 1968 H3N2 pandemic virus, induced cross-reactive immune responses against a recent 2005 H3N2 virus challenge. CONCLUSIONS: DNA vaccines based on pandemic or recent seasonal influenza genes induced cross-reactive immunity against contemporary virus challenge as good as or superior to contemporary conventional trivalent protein vaccines. This suggests a unique ability of influenza DNA to induce cross-protective immunity against both contemporary and long-time drifted viruses.

Aleutian mink disease virus and humans.

Reports of a possible relationship between Aleutian mink disease parvovirus (AMDV) and human infection are rare. However, 2 mink farmers with vascular disease and microangiopathy similar to that in mink with Aleutian disease were found to have AMDV-specific antibodies and AMDV DNA. These findings raise the suspicion that AMDV may play a role in human disease.

Nephropathy in type 1 diabetes is associated with increased circulating activated platelets and platelet hyperreactivity.

Patients with diabetes mellitus (DM) have increased platelet activation compared to non-diabetic controls. Platelet hyperreactivity has been associated with adverse cardiovascular outcomes in Type 2 DM, and with diabetic nephropathy. We investigated the relationship between platelet activation and nephropathy in Type 1 DM. Patients with Type 1 DM and diabetic nephropathy (n = 35), age- and sex-matched Type 1 DM patients with persistent normoalbuminuria (n = 51), and healthy age- and sex-matched controls (n = 30) were studied. Platelet surface P-selectin, platelet surface activated GPIIb/IIIa, monocyte-platelet aggregates (MPAs) and neutrophil-platelet aggregates (NPAs) were measured by whole blood flow cytometry as markers of platelet activation. Platelet reactivity was assessed in response to exogenously added ADP and thrombin receptor activating peptide (TRAP). Platelet surface P-selectin (basal and in response to 0.5 or 20 microM ADP) was higher in nephropathy patients compared with normoalbuminuric patients (P = 0.027), and non-diabetic controls (P = 0.0057). NPAs were higher in nephropathy patients compared to normoalbuminuric patients (P = 0.0088). MPAs were higher in nephropathy patients compared to non-diabetic controls (P = 0.0075). There were no differences between groups in activated GPIIb/IIIa or in response to TRAP at any end-point. More patients with nephropathy received aspirin (71.4%) compared to normoalbuminuric patients (27.4%) (P < 0.0001). Type 1 diabetic nephropathy, as compared with normoalbuminuria, is associated with circulating activated platelets and platelet hyperreactivity to ADP, despite the confounding variable of more nephropathy patients receiving aspirin. This platelet activation is likely to contribute to the known increased risk of cardiovascular events in patients with diabetic nephropathy.

Early life DNA vaccination with the H gene of Canine distemper virus induces robust protection against distemper.

Young mink kits (n=8) were vaccinated with DNA plasmids encoding the viral haemagglutinin protein (H) of a vaccine strain of Canine distemper virus (CDV). Virus neutralising (VN) antibodies were induced after 2 immunisations and after the third immunisation all kits had high VN antibody titres. The VN antibody titres remained high for more than 4 months and the mink were protected against viraemia, lymphopenia, clinical disease and changes in the percentage of IFN-gamma producing peripheral blood leucocytes after challenge inoculation with a recent wild type strain of CDV. Essentially, these results demonstrate that early life DNA vaccination with the H gene of a CDV vaccine strain induced robust protective immunity against a recent wild type CDV.

Characterization of antibodies against ferret immunoglobulins, cytokines and CD markers.

Ferret IgG and IgM were purified from normal serum, while ferret IgA was purified from bile. The estimated molecular weights of the immunoglobulin gamma, alpha and mu heavy chains were found to be 54kDa, 69kDa and 83kDa, respectively. For immunological (ELISA) quantification of ferret immunoglobulins, we identified and characterized polyclonal antibodies towards ferret IgG, IgM and IgA. We also identified 22 monoclonal antibodies (mAbs) raised mostly against human CD markers which cross-reacted with ferret leukocytes. These antibodies were originally specific against human CD8, CD9, CD14, CD18, CD25, CD29, CD32, CD44, CD61, CD71, CD79b, CD88, CD104, CD172a and mink CD3. Finally, we identified 4 cross-reacting mAbs with specificities against ferret interferon-gamma, TNF-alpha, interleukin-4 and interleukin-8.

Lymphotropism and host responses during acute wild-type canine distemper virus infections in a highly susceptible natural host.

The mechanisms behind the in vivo virulence of immunosuppressive wild-type morbillivirus infections are still not fully understood. To investigate lymphotropism and host responses, we have selected the natural host model of canine distemper virus (CDV) infection in mink. This model displays multisystemic infection, similar to measles virus and rinderpest virus infections in their susceptible natural hosts. The wild-type CDVs investigated provoked marked virulence differences, inducing mild versus marked to severe acute disease. The mildly virulent wild-type virus induced transient lymphopenia, despite the development of massive infection of peripheral blood mononuclear cells (PBMCs) exceeding that determined for the highly virulent wild-type virus, indicating an inverse relationship between acute virulence and the extent of viraemia in the investigated wild-type viruses. Single-cell cytokine production in PBMCs was investigated throughout the acute infections. We observed Th1- and Th2-type cytokine responses beginning in the prodromal phase, and late inflammatory responses were shared between the wild-type infections.

Humoral and cell-mediated immune responses in DNA immunized mink challenged with wild-type canine distemper virus.

The aim of the study was to investigate the different phases of the immune response after DNA immunization with the hemagglutinin and nucleoprotein genes from canine distemper virus (CDV). Although attenuated live CDV vaccines have effectively reduced the incidence of disease, canine distemper is still a problem worldwide. The broad host range of CDV creates a constant viral reservoir among wildlife animals. Our results demonstrated early humoral and cell-mediated immune responses (IFN-gamma) in DNA vaccinated mink compared to mock-vaccinated mink after challenge with a Danish wild-type CDV. The DNA vaccine-induced immunity protected the natural host against disease development.

Summary of the animal homologue section of HLDA8.

The development of reagents against leukocyte differentiation antigens in veterinary species is delayed compared to mouse and men and therefore also the number of existing reagents for the characterisation of leukocytes derived from species with importance in veterinary medicine is restricted. Cross-reactive studies with existing well defined monoclonal antibodies directed against leukocyte differentiation antigens derived from other species are an alternative approach to enhance the panel of reagents in veterinary immunology. This study describes the activities of the animal homologue section in frame of human leukocyte differentiation antigen 8-workshop (HLDA8) were 376 monoclonal antibodies, mainly directed against human leukocytes had been tested for their reactivity with 17 different animal species including non-human primates, ruminants, swine, horse, carnivores, rabbit, guinea pig, chicken and fish. In a first round 182 mAb were selected based on there reactivity in FCM analyses with at least one species for further studies, including multi-colour FCM, and molecular analyses of the antigens. Interesting was the species-overlapping reactivity of mAb directed against distinct clusters: 11 out of 17 species reacted with CD9, 11 of 17 with CD11a, CD14 (11/17), CD18 (13/17), CD21 (7/17), CD29 (10/17), CD44 (13/17), CD45 (9/17), CD47 (10/17), CD49d (13/17), CD61 (6/17), CD86 (7/17), CD91 (5/17), and CD172a (10/17), indicating evolutionary highly conserved epitopes on these surface molecules. Our results suggest the suitability of cross-reactive mAb for the animal model studies. Moreover, these findings contribute to our understanding of the evolution of the immune system.

Vaccination of mice with plasmids expressing processed capsid protein of foot-and-mouth disease virus--importance of dominant and subdominant epitopes for antigenicity and protection.

The capsid of foot-and-mouth disease virus (FMDV) displays several independent B cell epitopes, which stimulate the production of neutralising antibodies. Some of these epitopes are highly variable between virus strains, but dominate the immune response. The site A on VP1 is the most prominent example of a dominant and variable site. This variability is a problem when designing vaccines against this disease, because it necessitates a close match between vaccine strain and virus in an outbreak. We have introduced a series of mutations into viral capsid proteins with the aim of selectively silencing two dominant and highly variable epitopes and thereby divert immune responses toward less dominant but more conserved, protective epitopes. When mice were immunized with modified antigens, the resulting immune responses showed a higher degree of cross-reactivity towards heterologous virus as compared to mice vaccinated with wild type epitopes. Most of the modifications did not adversely affect the ability of the plasmids to induce complete protection of mice against homologous challenge.

Various domains of the B-cell regulatory molecule CD72 has diverged at different rates in mammals: cloning, transcription and mapping of porcine CD72.

We report the cloning of the porcine B-cell co-receptor CD72, as well as genomic mapping and examination of transcription. The B-cell receptor (BCR) complex mediates signalling upon antigen recognition by the membrane bound BCR. Several co-receptors modulate this signal positively or negatively. CD72 has been shown to be a negatively regulating BCR co-receptor. We isolated and sequenced three porcine CD72 transcript variants. Using a pig radiation hybrid panel we found the porcine CD72 gene to be located on chromosome 1q21-28 in a region syntenic to human chromosome 9. The porcine CD72 gene is highly transcribed in lymph node, thymus and lung tissues as well as in pulmonary alveolar macrophages. The predicted porcine CD72 polypeptide shows conservation of immunoreceptor tyrosine-based inhibitory motifs and an extracellular C-type lectin domain. Compared to CD72 sequences from other mammals as well as from chicken, the polypeptide is highly conserved in the intracellular part and much less conserved in the extracellular part. We suggest that this difference might be due to the different nature of ligands and the constrains on these to co-evolve.

Cytokine mRNA profiles in pigs exposed prenatally and postnatally to Schistosoma japonicum.

The pig is a natural host for Schistosoma japonicum and a useful animal model of human infection. The aim of the present study was to assess the differences between the cytokine profiles in prenatally or postnatally S. japonicum exposed pigs. Seven prenatally exposed pigs, 7 postnatally exposed pigs and 4 uninfected control pigs were compared 27 weeks post exposure. Variables included worm burdens, tissue egg counts, liver pathology and mRNA levels of IL-2, IL-4, IL-10, IL-12, TNF-alpha, TGF-beta1 and IFN-gamma in the liver and the caecum, assessed by RT-PCR. Infection intensity and level of septal fibrosis were significantly higher in the postnatal group compared to the prenatal group (P < 0.05). A significant increase of IL-4 (P < 0.01), IL-10 (P < 0.01), IL-12 (P < 0.01) and TNF-alpha (P < 0.05) mRNA level was also observed in the caecum of prenatally infected animals compared to the control group (P < 0.01). The prenatal group showed higher levels of TGF-beta1 in the liver compared with the postnatally infected group (P< 0.05) and the control group (P< 0.01). This suppressive immune response correlated with previously reported low hepatic pathogenesis in prenatally exposed pigs.

Cloning, characterization and mapping of porcine CD14 reveals a high conservation of mammalian CD14 structure, expression and locus organization.

The cell surface protein CD14 plays a central role in innate immunity as a pattern recognition receptor. CD14 is part of a receptor complex also including toll-like receptor 4 and MD2 proteins. Binding of the ligand lipopolysaccharide to the complex on myeloid cells leads to release of pro-inflammatory cytokines and mediators from the cell. In this study, we present the cloning, characterization and tissue expression pattern of a porcine CD14 encoding cDNA, and the chromosomal localization of the porcine CD14 gene. The open reading frame is predicted to encode a protein of 373 amino acids, which shows conservation of structural as well as functional regions when compared to other mammalian species. The CD14 gene was localized to porcine chromosome 2 in a region syntenic to human chromosome 5q. Transcription analysis shows that CD14 is widely expressed in tissues examined in this study, which correlates well with expression primarily on myeloid cells.

A time course study of immunological responses in Trichuris suis infected pigs demonstrates induction of a local type 2 response associated with worm burden.

In order to investigate immunological changes over time in pigs infected with Trichuris suis, we inoculated 40 pigs with 5000 infective T. suis eggs and left 40 pigs as uninfected controls. Equal numbers of pigs from both groups were sacrificed every other week from 1 to 11 weeks p.i. At necropsy tissue samples were collected from all pigs and their worm burdens were determined. In the proximal colon of T. suis-infected pigs infiltration of eosinophils peaked 5 weeks p.i. and mast cell infiltration developed from 5 to 11 weeks p.i. Histological evaluation of the proximal colon revealed that the presence of T. suis was closely associated with intestinal histopathological changes such as crypt hyperplasia, goblet cell hyperplasia and a general hypertrophy of mucosa. The crypt lengths were positively associated with worm burdens. Real-time PCR analysis of genes related to immune function indicate a local increased transcription of genes coding for CCR3, ARG1, MUC5AC, IL-4, IL-5, IL-13, FcepsilonR1alpha, and IL-13Ralpha2 and decreased expression of genes coding for iNOS, TNF-alpha, IL-10, CD3epsilon, CD80, CD86, IL-4Ralpha, IL-13Ralpha1 and CD40 in the proximal colon of pigs infected with T. suis. This local T-helper cell Type 2-like gene-expression pattern indicates that the Type 2 immune response characteristic of helminth infections in both mouse and humans also develops in pigs infected with T. suis. The results from this study expand our knowledge of the immunomodulatory effect of T. suis, a parasite that has proven effective in treating inflammatory bowel disease, when its eggs are administered regularly to patients.